PROJECT SUMMARY/ABSTRACT Cutaneous T-cell lymphoma (CTCL) is a rare, but potentially devastating malignancy of the skin. Early detection of CTCL is associated with positive clinical outcomes, but diagnosis is often complicated by the heterogeneous clinical and histological presentation. Additionally, systemic therapies remain sub- optimal for CTCL, highlighting the need for new treatment strategies. To date, most studies have been performed on blood from CTCL patients, which has revealed a complex interplay between host immunity, malignant T-cells and the tumor microenvironment. However, the molecular mechanisms that govern disease pathogenesis, especially in the skin, remain largely undefined. The goal of the proposed research is to use new technologies that allow for multi-parametric, high- resolution analyses in formalin-fixed paraffin-embedded (FFPE) skin tissue samples to define the immune and metabolic profiles of CTCL at different stages of disease. The first technology is Multiplexed Ion Beam Imaging (MIBI), which uses secondary ion mass spectrometry to visualize up to 100 metal conjugated antibodies simultaneously at nanometer resolution in FFPE tissue sections. Specifically, Aim I will use MIBI to define the heterogeneity and plasticity of cells that exist at the CTCL tumor-host interface, to improve the precision with which CTCL is diagnosed and treated. The second technology is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), which can detect thousands of metabolites in FFPE tissue sections. Specifically, Aim II will use MALDI-MSI to determine the reflective metabolic activity of the CTCL microenvironment, thereby adding functional insight to disease pathogenesis and revealing potential metabolically active therapeutic targets. Overall, these studies will characterize the molecular heterogeneity and functional complexity of CTCL, which will shed light on how this disease evades immune destruction and reprograms energy- metabolism. As such, this work promises to improve the detection, treatment and clinical outcomes for CTCL.